Construction method of patrinia characteristic spectrum and contrast spectrum and method for distinguishing patrinia from counterfeit products

Abstract

The invention discloses a construction method of a patrinia characteristic spectrum and a reference spectrum, and a method for distinguishing patrinia and counterfeit products. The construction method of the specific chromatogram comprises the following steps: respectively sucking a reference substance solution and a test sample solution for ultra-high performance liquid chromatography analysis, and constructing the specific chromatogram of a sample to be detected, the specific chromatogram comprising at least 14 characteristic peaks, four characteristic peaks in the characteristic peaks respectively correspond to a protocatechuic acid reference substance peak, a rhodamine reference substance peak, a 3, 4-O-dicaffeoylquinic acid reference substance peak and a 4, 5-O-dicaffeoylquinic acid reference substance peak; and carrying out qualitative analysis on the to-be-detected sample through the obtained characteristic chromatogram, and simultaneously identifying the patrinia scabiosaefolia, the patrinia villosa juss and the counterfeit patrinia herb. The method is simple to operate, and methodology verifies that the reproducibility and stability are good, the detection cost is low, and the detection efficiency is high.

Description

Technical Field

[0001] The present invention relates to a method for constructing a drug characteristic spectrum and an application thereof, and in particular to a method for constructing a Patrinia chinensis characteristic spectrum, a reference spectrum, and a method for distinguishing Patrinia chinensis from counterfeit products. Background Art

[0002] Patrinia scabiosaefolia Fisch. or Patrinia villosa Juss. is a plant of the Patriniaceae family. It is pungent and bitter in taste, slightly cold in nature, and has the effects of clearing away heat and detoxifying, eliminating carbuncle and draining pus, removing blood stasis and relieving pain. It is used to treat appendicitis, dysentery, enteritis, hepatitis, abdominal pain caused by postpartum blood stasis, carbuncle, furuncle, etc. (China Pharmacopoeia Committee. Pharmacopoeia of the People's Republic of China. Volume 1 [S]. Beijing: China Medical Science and Technology Press, 1977: 349-350.).

[0003] Patrinia salsa is a traditional Chinese medicine, first recorded in Shennong's Herbal Classic. Studies have shown that the main chemical components of Patrinia salsa include triterpenoid saponins, cyclopentane ether terpenes, flavonoids, phenylpropanoids, volatile oils and organic acids (Chen Shuling, Han Liang. Modern research progress on Patrinia salsa [J]. Journal of Guangdong Pharmaceutical University, 2017, 33(06): 816-821.). Pharmacological studies have shown that Patrinia salsa has antiviral (Xiao Hehe, Liu Mingzhu, Yu Qing, et al. Antiviral effect of Patrinia salsa water extract on grouper iridovirus [J]. Journal of Guangxi Academy of Sciences, 2019, 35(03): 185-192.) and anti-inflammatory (Sun Dajuan, Wei Xiunan, Cheng Yan, et al. Effect of Astragalus-Paradise herbal medicine pair on JAK1 / STAT6 / SOCS1 signaling pathway in HT-29 cell inflammation model [J]. Journal of Traditional Chinese Medicine, 2023, 64(03): 295-302.), anti-tumor (Zhang Pingping, Jia Ru, Cen Rong, et al. Research progress on the mechanism of action of flavonoids in Patrinia salsa on the occurrence and development of colorectal cancer [J]. Chinese Journal of Experimental Traditional Chinese Medicine, 2024, 30(04): 240-247.), liver protection (Li Aichen, Fang Lei. Protective effect of Patrinia salsa polysaccharide on liver tissue of rats with exercise fatigue [J]. Food Research and Development, 2016, 37(12): 174-177.) and other pharmacological activities.

[0004] Chinese medicine formula granules are made from single Chinese medicine slices through water extraction, concentration, drying and granulation. They are a new form of slice reform. Compared with traditional slice decoctions, they are easy to carry and simple to use, and can meet the requirements of modern fast-paced work and life. In recent years, Chinese medicine formula granules have developed rapidly, and national standards for formula granules of multiple Chinese medicine varieties have been issued one after another, which has made great contributions to standardizing the production of Chinese medicine formula granules and promoting the modernization of Chinese medicine. Patrinia scabra formula granules are made from Patrinia scabra slices through modern technology extraction, concentration, drying and granulation, which retains the clinical effects of Patrinia scabra while taking into account the portability of use.

[0005] At present, there are few reports on the characteristic spectrum of Patrinia salsa and the identification of mixed and counterfeit products. Zhang Lei et al. studied the microscopic identification characteristics and HPLC fingerprint spectrum of Patrinia salsa and its mixed and counterfeit products. The results showed that there were significant differences in the microscopic characteristics of the medicinal powder of Patrinia salsa and its mixed and counterfeit products, and there were also differences in the total saponin content. A total of 26 batches of HPLC fingerprints of Patrinia salsa and its mixed and counterfeit products were established, and 11 common peaks were calibrated. Through similarity analysis, cluster analysis and principal component analysis, Patrinia salsa and its mixed and counterfeit products can be accurately distinguished (Zhang Lei, Huang Ziru, Lin Ziyi, et al. Study on the microscopic identification characteristics and HPLC fingerprint spectrum of Patrinia salsa and its mixed and counterfeit products [J]. Chinese Herbal Medicine, 2024, 55(06): 2066-2076.). Yu Kunzi et al. established an identification method for Herba Patriniae and its counterfeits by means of property identification and microscopic identification, and conducted a comparative study on Herba Patriniae and its counterfeits on the market. They can be distinguished from each other by property characteristics such as stem surface and cross-section characteristics, stem leaf morphology, inflorescence hair condition, odor, and microscopic characteristics such as stem cross section (Yu Kunzi, Han Shikai, Ma Siyu, et al. Pharmacognosy research on Herba Patriniae [J]. Journal of Drug Analysis, 2024, 44(07): 1255-1266.). Wang Yinglan et al. used high performance liquid chromatography (HPLC) to study the fingerprint of Patrinia scabra and obtained the HPLC fingerprint of Patrinia scabra. At the same time, the key components of chlorogenic acid and rutin were determined, and 11 common peaks were calibrated as key indicators for the quality evaluation of Patrinia scabra (Wang Yinglan, Liu Yuankun, Wan Haiqing, et al. Establishment and optimization of quality standards for Patrinia scabra [J]. Clinical Rational Drug Use, 2024, 17(17): 154-157.). Liu Suxiang et al. established the HPLC fingerprint of Patrinia scabra and calibrated 8 common peaks. Cluster analysis showed that the similarity between the fingerprint of Class I medicinal materials and the reference was 0.987-0.907, and the difference between the fingerprint of Class II medicinal materials and the reference was large. It can be used as an effective means to evaluate the quality of Patrinia scabra from different origins and varieties (Liu Suxiang, Liu Yi, Bai Xue, et al. Study on fingerprint of Patrinia scabra [J]. Chinese Herbal Medicine, 2016, 47(12): 2074-2077.). According to the existing literature, the quality control research of Patrinia scabra is mainly focused on medicinal materials, and there are fewer research materials on the quality control of Patrinia scabra water extract. Through patent retrieval, the patent No. CN111208253A uses high performance liquid chromatography-mass spectrometry to detect Patrinia scabra components, establishes Patrinia scabra HPLC fingerprint, establishes Patrinia scabra control fingerprint common mode through system cluster analysis and similarity evaluation system, and judges the quality of medicinal materials. This invention patent does not carry out the different bases and authenticity identification research of Patrinia scabra, and the quality evaluation research is not comprehensive enough, and the quality level of Patrinia scabra cannot be effectively reflected. The characteristic spectrum method constructed by the patent of the present invention can simultaneously identify the different bases and authenticity of Patrinia scabra, can comprehensively control the quality of medicinal materials, and ensure the effectiveness and safety of product quality. Summary of the invention

[0006] Purpose of the invention: The present invention aims to establish a characteristic spectrum of Patrinia salsa with good reproducibility, stability, low detection cost and high detection efficiency, a method for constructing a reference spectrum, and a method for distinguishing Patrinia salsa from counterfeits, which can quickly and accurately identify Patrinia salsa with yellow flower, Patrinia salsa with white flower and Patrinia salsa counterfeits, provide a theoretical basis for the quality control of Patrinia salsa, and provide a more powerful guarantee for the industrialization of Patrinia salsa formula granules.

[0007] Technical solution: The present invention provides a method for constructing a characteristic map of Patrinia scabra, comprising the following steps:

[0008] (1) Take the powder of Patrinia lappa herb, intermediate, decoction piece, standard decoction or its formula granules respectively, add water to dissolve, extract with n-butanol, evaporate the extract to dryness, and add methanol aqueous solution to dissolve the residue to prepare the test solution;

[0009] (2) respectively taking protocatechuic acid, choline glycoside, 3,4-O-dicaffeoylquinic acid, and 4,5-O-dicaffeoylquinic acid, and adding solvent to prepare reference substance solutions;

[0010] (3) The test solution and the reference solution are injected into an ultra-high performance liquid chromatography respectively to obtain a characteristic spectrum; the characteristic spectrum has 14 common peaks, wherein protocatechuic acid is taken as the reference, and peak 1 is the chromatographic peak of protocatechuic acid; quinoline is taken as the reference, and peak 3 is the chromatographic peak of quinoline; 3,4-O-dicaffeoylquinic acid is taken as the reference, and peak 7 is the chromatographic peak of 3,4-O-dicaffeoylquinic acid; 4,5-O-dicaffeoylquinic acid is taken as the reference, and peak 10 is the chromatographic peak of 4,5-O-dicaffeoylquinic acid; the peak corresponding to the quinoline reference is the S peak, and the relative retention time of the remaining peaks and the S peak is calculated. Their relative retention times should be within ±10% of the specified values. The specified values ​​of Peak 2, Peaks 4-6, Peak 8, Peak 9, and Peaks 11-14 are 0.55, 1.05, 1.07, 1.17, 1.82, 1.94, 2.34, 2.51, 2.54, and 2.61, respectively.

[0011] The chromatographic conditions of the ultra-high performance liquid chromatography are as follows: GOLD aQ is used as a chromatographic column; methanol is used as a mobile phase A, and a 0.1% formic acid solution is used as a mobile phase B; the flow rate is 0.23-0.27 ml / min; the column temperature is 25-30° C.; the detection wavelength is 254 nm; the gradient elution is 0-3 min, the volume fraction of the mobile phase A is 10%, and the volume fraction of the mobile phase B is 90%; 3-22 min, the volume fraction of the mobile phase A changes from 10 to 26%, and the volume fraction of the mobile phase B changes from 90 to 74%; 22-34 min, the volume fraction of the mobile phase A changes from 26 to 36%, and the volume fraction of the mobile phase B changes from 74 to 64%; 34-55 min, the volume fraction of the mobile phase A changes from 36 to 56%, and the volume fraction of the mobile phase B changes from 64 to 44; 55-58 min, the volume fraction of the mobile phase A is 56%, and the volume fraction of the mobile phase B is 44%.

[0012] The extraction times in step (1) are 1 to 3 times, and the extraction method is shaking.

[0013] Preferably, 0.2 g to 1 g of Patrinia scabra granules are taken, ground, dissolved in water, extracted 1 to 3 times with n-butanol by shaking, the extract is evaporated to dryness, and the residue is dissolved in methanol aqueous solution to serve as the test solution.

[0014] Preferably, 1 g to 3 g of Patrinia patriniae reference medicinal material is taken, water is added, the mixture is decocted on an electric stove, filtered, the filtrate is extracted 1 to 3 times by shaking with n-butanol or ethyl acetate, the extract is evaporated to dryness, and the residue is dissolved in methanol aqueous solution as a reference medicinal material reference solution; separately, appropriate amounts of protocatechuic acid reference substance, chondrin reference substance, 3,4-O-dicaffeoylquinic acid reference substance, and 4,5-O-dicaffeoylquinic acid reference substance are taken, dissolved in methanol aqueous solution as reference substance reference solution.

[0015] Preferably, 0.5 μl to 2 μl of the reference solution and the test solution are respectively pipetted.

[0016] The re-dissolving solvent is a methanol aqueous solution with a volume concentration of 10% to 70%.

[0017] Wherein, in step (1), the concentration of the protocatechuic acid reference substance solution is 15 to 40 μg / ml; the concentration of the glycoside reference substance solution is 100 to 200 μg / ml; the concentration of the 3,4-O-dicaffeoylquinic acid reference substance solution is 15 to 40 μg / ml; and the concentration of the 4,5-O-dicaffeoylquinic acid reference substance solution is 15 to 40 μg / ml.

[0018] The present invention also provides a method for constructing a reference map of Patrinia scabra, comprising the following steps:

[0019] (1) preparing multiple batches of Patrinia chinensis herb test solution, Patrinia chinensis intermediate test solution, Patrinia chinensis decoction piece test solution, Patrinia chinensis standard decoction test solution or Patrinia chinensis formula granule test solution;

[0020] (2) According to the method for constructing the characteristic spectrum of Patrinia chinensis, the test solution is tested respectively to obtain the characteristic spectrum of Patrinia chinensis medicinal materials, the characteristic spectrum of intermediates, the characteristic spectrum of decoction pieces, the characteristic spectrum of standard decoction and the characteristic spectrum of formula granules;

[0021] (3) The obtained characteristic spectrum is introduced into the Chinese medicine chromatographic fingerprint spectrum similarity evaluation system to establish a reference spectrum of Patrinia scabra medicinal materials, intermediates, decoction pieces, standard decoctions or their formula granules.

[0022] The present invention also provides a method for distinguishing Patrinia lutea from Patrinia odorata, comprising the following steps:

[0023] (1) Take the powder of the medicinal material, intermediate, decoction piece, standard decoction or its formula granules of Patrinia chrysantha or Patrinia dasyphylla respectively, add water to dissolve it, extract it with n-butanol, evaporate the extract to dryness, and add methanol-water solution to dissolve the residue to prepare the test solution;

[0024] (2) respectively taking protocatechuic acid, choline glycoside, 3,4-O-dicaffeoylquinic acid, and 4,5-O-dicaffeoylquinic acid, and adding solvent to prepare reference substance solutions;

[0025] (3) The test sample solution and the reference sample solution were injected into an ultra-high performance liquid chromatography to obtain characteristic spectra; by analyzing the relative peak areas of peaks 8 and 11 relative to peak 3 in Patrinia chinensis, Patrinia chrysantha and Patrinia leucoderma can be distinguished; when the relative peak area of ​​peak 11 is greater than 0.24 and the relative peak area of ​​peak 8 is greater than 0.30, the test sample is Patrinia chrysantha; when peak 11 is less than 0.24 and peak 8 is less than 0.30, the test sample is Patrinia leucoderma.

[0026] The chromatographic conditions of the ultra-high performance liquid chromatography are as follows: GOLD aQ is used as a chromatographic column; methanol is used as a mobile phase A, and a 0.1% formic acid solution is used as a mobile phase B; the flow rate is 0.23-0.27 ml / min; the column temperature is 25-30° C.; the detection wavelength is 254 nm; the gradient elution is 0-3 min, the volume fraction of the mobile phase A is 10%, and the volume fraction of the mobile phase B is 90%; 3-22 min, the volume fraction of the mobile phase A changes from 10 to 26%, and the volume fraction of the mobile phase B changes from 90 to 74%; 22-34 min, the volume fraction of the mobile phase A changes from 26 to 36%, and the volume fraction of the mobile phase B changes from 74 to 64%; 34-55 min, the volume fraction of the mobile phase A changes from 36 to 56%, and the volume fraction of the mobile phase B changes from 64 to 44; 55-58 min, the volume fraction of the mobile phase A is 56%, and the volume fraction of the mobile phase B is 44%.

[0027] The present invention also provides a method for distinguishing Patrinia lutea from counterfeit products, comprising the following steps:

[0028] (1) Take the powder of Patrinia chrysanthemi or counterfeit medicinal materials, intermediates, decoction pieces, standard decoction or its formula granules respectively, add water to dissolve, extract with n-butanol, evaporate the extract to dryness, and add methanol-water solution to dissolve the residue as the test solution;

[0029] (2) respectively taking protocatechuic acid, choline glycoside, 3,4-O-dicaffeoylquinic acid, and 4,5-O-dicaffeoylquinic acid, and adding solvent to prepare reference substance solutions;

[0030] (3) The test sample solution and the reference sample solution were injected into an ultra-high performance liquid chromatography to obtain characteristic spectra; by analyzing the relative peak areas of peaks 8 and 11 in Patrinia scabra relative to peak 3, Patrinia scabra and its counterfeits, Herba Epimedii and Herba Cynoglossi, could be distinguished; when the relative peak area of ​​peak 11 was greater than 0.24 and the relative peak area of ​​peak 8 was greater than 0.30, the test sample was Patrinia scabra; when peaks 11 and 8 were not detected, the test sample was Herba Cynoglossi; when peak 3 was not detected, the test sample was Herba Cynoglossi.

[0031] Among them, the preparation of the reference medicinal material reference solution: take 1g to 3g of Patrinia scabra reference medicinal material, put it in a stoppered conical flask, add water, decoct on an electric stove, filter, and extract the filtrate by shaking with n-butanol or ethyl acetate 1 to 3 times, evaporate the extract, and add methanol aqueous solution to dissolve the residue as the reference medicinal material solution.

[0032] Preparation of reference substance solution: Take appropriate amount of protocatechuic acid reference substance, dapoxetine reference substance, 3,4-O-dicaffeoylquinic acid reference substance, and 4,5-O-dicaffeoylquinic acid reference substance, accurately weigh them, add 10% to 70% methanol to make each ml contain 15 to 40 μg of protocatechuic acid; 100 to 200 μg of dapoxetine; 15 to 40 μg of 3,4-O-dicaffeoylquinic acid; and 15 to 40 μg of 4,5-O-dicaffeoylquinic acid, as reference substance solution.

[0033] Preparation of the test solution of the Patrinia scabra granules: take about 0.3 g of Patrinia scabra granules powder, place it in a stoppered conical flask, add 20 ml of water to dissolve it, shake and extract with n-butanol twice, 20 ml each time, combine the n-butanol solutions, evaporate to dryness, add 2 ml of 50% methanol to dissolve the residue, and use it as the test solution.

[0034] Preparation of test solution of standard decoction of Patrinia salviae jasminoides (Painted with yellow flower, Patrinia leucophylla) and its counterfeit products (Painted with calendula officinalis, Cyperus rotundus): Take about 0.2-1.0 g of standard decoction powder, place it in a stoppered conical flask, add water to dissolve it, shake and extract with n-butanol or ethyl acetate for 1-3 times, evaporate the extract to dryness, add methanol-water solution to dissolve the residue, and use it as the test solution.

[0035] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0036] (1) The method of the present invention is a simpler, faster and more effective method for detecting the characteristic spectrum of Patrinia patriniae formula granules, which can simultaneously identify different origins and their counterfeits, providing a more powerful guarantee for the industrialization of Patrinia patriniae formula granules;

[0037] (2) The characteristic spectrum method established by the present invention can simultaneously distinguish different origins of Patrinia scabra and their counterfeits, providing a reliable analytical method for identifying different origins of Patrinia scabra and their counterfeits. The method is simple to operate and the results are relatively objective and accurate. After methodological verification, it has good reproducibility and stability, low detection cost and high detection efficiency, which provides an important guarantee for the standardization of the production of Patrinia scabra formula granules.

[0038] (3) The characteristic spectrum method established by the method of the present invention presents rich chromatographic peak information, including multiple chemical components, a large number of characteristic peaks, and 14 common characteristic peaks. Some characteristic peaks are identified, which is more than the number of characteristic peaks specified in the published standard of Patrinia scabra formula granules, and the evaluation is more comprehensive; and the separation of each characteristic peak is good and the baseline is stable. At the same time, the use of control medicinal materials as reference can better reflect the consistency of the formula granules and the medicinal materials, and can more comprehensively control the quality of the Patrinia scabra formula granules;

[0039] (4) The chromatographic peak information presented in the characteristic spectrum is rich, including multiple chemical components such as iridoids, phenolic acids, phenylpropanoids, and flavonoid glycosides, which can more comprehensively reflect the quality of Patrinia scabra.

[0040] (5) The present invention adopts ultra-high liquid chromatography and reasonably optimizes the chromatographic conditions to establish the characteristic spectrum of the Patrinia scabra formula granules, and simultaneously distinguishes different bases and authenticity, providing important guarantee for the quality control of the raw materials of the Patrinia scabra formula granules. The method is simple to operate, has high detection sensitivity, and low detection time and cost, and provides a new analytical means for the quality control of the Patrinia scabra formula granules. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 UPLC chromatograms of Patrinia scabra granules at different detection wavelengths;

[0042] Figure 2 UPLC diagrams of different extraction methods and solvents;

[0043] Figure 3 UPLC diagrams for different extraction times;

[0044] Figure 4 The chromatograms of the reference substance, reference medicinal material, and Patrinia scabra granules;

[0045] Figure 5 This is the characteristic spectrum of the Patrinia scabra granules, peak 1: protocatechuic acid; peak 3 (S): danshinone; peak 7: 3,4-O-dicaffeoylquinic acid; peak 10: 4,5-O-dicaffeoylquinic acid;

[0046] Figure 6 This is the characteristic spectrum of the intermediate of Patrinia scabra, peak 1: protocatechuic acid; peak 3 (S): danshin; peak 7: 3,4-O-dicaffeoylquinic acid; peak 10: 4,5-O-dicaffeoylquinic acid;

[0047] Figure 7 This is the characteristic spectrum of the standard decoction of Patrinia scabra, peak 1: protocatechuic acid; peak 3 (S): danshin; peak 7: 3,4-O-dicaffeoylquinic acid; peak 10: 4,5-O-dicaffeoylquinic acid;

[0048] Figure 8 This is the characteristic spectrum of Patrinia scabra decoction pieces, peak 1: protocatechuic acid; peak 3 (S): danshinone; peak 7: 3,4-O-dicaffeoylquinic acid; peak 10: 4,5-O-dicaffeoylquinic acid;

[0049] Fig. 9 This is the characteristic spectrum of Patrinia scabra. Peak 1: protocatechuic acid; Peak 3 (S): danshin; Peak 7: 3,4-O-dicaffeoylquinic acid; Peak 10: 4,5-O-dicaffeoylquinic acid;

[0050] Fig.10 This is a test on the specificity of the Patrinia scabra granules;

[0051] Fig.11 This is a test on the integrity of the Patrinia scabra formula granules;

[0052] Fig.12 To investigate the chromatographic column for the characteristic spectrum of Patrinia scabra granules;

[0053] Fig.13 The column temperature survey of the characteristic spectrum of Patrinia scabra granules;

[0054] Fig.14 It is a flow rate investigation of the characteristic spectrum of Patrinia scabra formula granules;

[0055] Fig.15 It is a comparison of the characteristic spectra of different standard decoctions of Patrinia scabra.

[0056] Fig.16 This is a comparison of the characteristic spectra of the standard decoctions of authentic and fake Patrinia scabra. DETAILED DESCRIPTION

[0057] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0058] 1. Instruments and reagents

[0059] Agilent Technologies 1290 Inftnity ultra-high performance liquid chromatograph; Thermo Vanquish ultra-high performance liquid chromatograph; KQ-250B ultrasonic cleaning machine (Kunshan Ultrasonic Instrument Co., Ltd.); electronic analytical balance (Mettler-Toledo Instrument (Shanghai) Co., Ltd.); temperature-controlled water bath (Nantong Huatai Experimental Instrument Co., Ltd.); HY-4 oscillator (Jintan Kexing Instrument Factory); pure water system (Millipore); AS165W centrifuge (Azov (Shanghai) Trading Co., Ltd.); acetonitrile (chromatographic grade, Thermo Fisher); methanol (chromatographic grade, Thermo Fisher); formic acid (chromatographic grade, Aladdin); ultrapure water; other reagents were of analytical grade.

[0060] Protocatechuic acid reference substance, dapoxetine reference substance, and 4,5-O-dicaffeoylquinic acid reference substance were purchased from the China Food and Drug Inspection Institute with batch numbers of 110809-201906, 111742-200501, and 111894-202205, respectively, and the purity was 97.7%, 100%, and 94.9%, respectively. 3,4-O-dicaffeoylquinic acid reference substance was purchased from Chengdu Pusi Biotechnology Co., Ltd. with batch number PS001054 and a purity of 98.0%.

[0061] The reference drug Patrinia chinensis (Palmlea corylifolia) was purchased from China Food and Drug Inspection Institute with batch number 121271-201201.

[0062] Patrinia scabra (Palmlea corylifolia) formula granules (batch numbers: KL1129, KL1139, KL1149) were provided by Jiangyin Tianjiang Pharmaceutical Co., Ltd.

[0063] Patrinia scabra (Patrimonium wilfordii) standard decoction (batch number: DG2019, DG2020, DG2022, DG2023, DG2024, DG2027, DG2028, DG2029, DG3037, DG3039, DG3040, DG3043, DG3044, DG3045, DG3650, DG3651, DG3652 652), Patrinia scabra (white flower Patrinia scabra) standard decoction (batch numbers: DG6028, DG6029, DG6030), Patrinia scabra counterfeit Mu Tou Hui standard decoction (batch numbers: DG6047, DG6050, DG6052), Patrinia scabra counterfeit Cynanchum wilfordii standard decoction (batch numbers: DG6053, DG6054, DG6055) were provided by Jiangyin Tianjiang Pharmaceutical Co., Ltd.

[0064] Patrinia scabra (yellow flower Patrinia scabra) intermediates (batch numbers: ZJT1129, ZJT1139, ZJT1149), Patrinia scabra (yellow flower Patrinia scabra) slices (batch numbers: YP2019, YP2020, YP2022, YP2023, YP2024, YP2027, YP2028, YP2029, YP3037, YP3039, YP3040, YP3043, YP3044, YP3045, YP3650, Y P3651, YP3652), Patrinia scabra (Palmaceae) (batch numbers: YC2019, YC2020, YC2022, YC2023, YC2024, YC2027, YC2028, YC2029, YC3037, YC3039, YC3040, YC3043, YC3044, YC3045, YC3650, YC3651, YC3652) were provided by Jiangyin Tianjiang Pharmaceutical Co., Ltd.

[0065] Example 1 Establishment of characteristic spectrum method of Patrinia patriniae formula granules

[0066] This example is used to confirm the optimal solution for establishing the characteristic spectrum of Patrinia scabra samples, specifically including comparison of chromatographic conditions, wavelength selection, and sample solution preparation schemes, and selecting the optimal chromatographic conditions and sample preparation scheme.

[0067] 1. Determination of detection wavelength

[0068] Take an appropriate amount of Patrinia scabra (Painted with yellow flower) formula granules (batch number: KL1129), grind it into powder, take 0.3g, put it in a stoppered conical flask, add 20ml of water to dissolve it, shake and extract it with n-butanol twice, 20ml each time, combine the n-butanol solution, evaporate to dryness, add 2ml of 50% methanol solution to the residue to dissolve it as the test solution, accurately pipette 1μl of each test solution, inject it into the ultra-high performance liquid chromatograph, and record the chromatograms at different wavelengths (235nm, 300nm, 254nm), such as Figure 1 As shown. The chromatographic conditions are as follows: GOLD aQ (Thermo, 2.1×150 mm, 1.9 μm) as the chromatographic column; methanol as the mobile phase A, 0.1% formic acid solution as the mobile phase B, gradient elution as specified in Table 1; flow rate of 0.25 ml per minute; column temperature of 25°C.

[0069] Table 1

[0070]

[0071] The results showed that the baseline of the Herba Patriniae granules was stable at 254nm wavelength, the chromatographic peak information was rich and the response was good, so 254nm was finally selected as the detection wavelength.

[0072] In summary, the chromatographic conditions were determined as follows: GOLD aQ (Thermo, 2.1×150 mm, 1.9 μm) was used as the chromatographic column; methanol was used as the mobile phase A, 0.1% formic acid solution was used as the mobile phase B, and gradient elution was performed according to the provisions in Table 1; the flow rate was 0.25 ml per minute; the column temperature was 25°C; and the detection wavelength was 254 nm.

[0073] 2. Preparation of test solution

[0074] 2.1 Investigation of different extraction methods and solvents

[0075] Take an appropriate amount of Patrinia scabra (Painted Saposhnikovia divaricata) granules (batch number: KL1129), grind them into powder, take 0.3g, and place three times in parallel in stoppered conical flasks. Add 20ml of 50% methanol solution to one portion, and ultrasonically treat (power 250W, frequency 40kHz) for 30 minutes, let cool, shake well, filter, and take the filtrate to obtain; add 20ml of water to the other two portions to dissolve, shake and extract twice with n-butanol, and shake and extract twice with ethyl acetate, 20ml each time, combine n-butanol and ethyl acetate respectively, evaporate to dryness, and add 2ml of 50% methanol solution to the residue to dissolve it as the test solution. Accurately aspirate 1μl of each test solution, inject it into the ultra-high performance liquid chromatograph, measure it according to the above chromatographic conditions, and calculate the ratio of the characteristic peak area / sample weight. The results are shown in Table 2. Figure 2 .

[0076] Table 2 Comparison of extraction efficiency of different extraction methods and solvents (peak area / sample weight)

[0077]

[0078]

[0079] The results showed that the chromatographic peak information of the sample extracted by shaking with n-butanol was rich, the response was high and the separation was good, so n-butanol shaking extraction was finally selected.

[0080] 2.2 Investigation of different extraction times

[0081] Take an appropriate amount of Patrinia scabra (Painted with yellow flower) formula granules (batch number: KL1129), grind it into powder, take 0.3g, place it in three parallel portions, put it in a stoppered conical bottle, add 20ml of water to dissolve it, shake and extract it with n-butanol 1, 2 and 3 times, 20ml each time, combine the n-butanol solution, evaporate to dryness, add 2ml of 50% methanol solution to the residue to dissolve it, and use it as the test solution. Accurately take 1μl of each test solution, inject it into the ultra-high performance liquid chromatograph, measure it according to the above chromatographic conditions, calculate the characteristic peak area / sample weight, and the results are shown in Table 3. Figure 3 .

[0082] Table 3 Comparison of extraction efficiency of different extraction times (peak area / sample weight)

[0083]

[0084] The results showed that the number of chromatographic peaks obtained with different extraction times was consistent. As the number of extractions increased, the response of some chromatographic peaks increased. The response of each chromatographic peak after two shaking extractions was good. In order to save extraction time and cost, two extractions were finally selected.

[0085] 2.3 Determination of the preparation method of the test solution

[0086] According to the above research results, the method for preparing the test solution of the characteristic spectrum of Patrinia scabra granules is determined as follows:

[0087] Take an appropriate amount of Patrinia scabra granules, grind them into powder, take 0.3g, put it in a stoppered conical flask, add 20ml of water to dissolve it, shake and extract with n-butanol twice, 20ml each time, combine the n-butanol liquids, evaporate to dryness, add 2ml of 50% methanol to dissolve the residue, and use it as the test solution.

[0088] Example 2

[0089] Take 3 batches of Patrinia scabra (Huanghua Patrinia scabra) formula granules, 17 batches of Patrinia scabra (Huanghua Patrinia scabra) standard decoctions, 17 batches of Patrinia scabra (Huanghua Patrinia scabra) decoction pieces, 17 batches of Patrinia scabra (Huanghua Patrinia scabra) medicinal materials, 3 batches of Patrinia scabra (Huanghua Patrinia scabra) intermediates, 3 batches of Baihua Patrinia scabra standard decoctions, 3 batches of Mutouhui (Parinia scabra fake) standard decoctions, and 3 batches of Ipomoea acuta (Parinia scabra fake) standard decoctions as Patrinia scabra samples, and perform determination according to the following steps respectively:

[0090] (1) Select a sample of Patrinia chinensis and prepare a test solution; use Patrinia chinensis as a control medicinal material reference to prepare a control medicinal material reference solution; use protocatechuic acid, arachidyl glycoside, 3,4-O-dicaffeoylquinic acid and 4,5-O-dicaffeoylquinic acid as reference substances to prepare reference substance solutions respectively.

[0091] Among them, the preparation of the reference medicinal material reference solution: take 2g of Patrinia scabra reference medicinal material, put it in a stoppered conical flask, add 50ml of water, boil it on an electric stove for 30 minutes, cool, shake well, filter, and extract the filtrate by shaking with n-butanol twice, 50ml each time, combine the n-butanol liquids, evaporate to dryness, and add 1ml of 50% methanol to the residue to dissolve it as the reference medicinal material solution.

[0092] Preparation of reference substance solution: Take appropriate amount of protocatechuic acid reference substance, 3,4-O-dicaffeoylquinic acid reference substance, 4,5-O-dicaffeoylquinic acid reference substance and glycoside reference substance, weigh accurately, add 50% methanol to make a mixed solution containing 30 μg of protocatechuic acid, 3,4-O-dicaffeoylquinic acid, 4,5-O-dicaffeoylquinic acid and 150 μg of glycoside per 1 ml, as the reference substance solution.

[0093] Preparation of test solution of Herba Patriniae Formula Granules: Take an appropriate amount of Herba Patriniae Formula Granules, grind them into powder, take 0.3 g, put it in a stoppered conical flask, add 20 ml of water to dissolve it, shake and extract with n-butanol twice, 20 ml each time, combine the n-butanol solutions, evaporate to dryness, add 2 ml of 50% methanol to dissolve the residue, and use it as the test solution.

[0094] Preparation of test solutions of standard decoctions of Patrinia salviae (Papilioides alba, Patrinia dasyphyllae), fake Patrinia salviae (Moutouhui, Cyperus rotundus), and intermediates of Patrinia salviae (Papilioides alba): Take 0.3 g of the standard decoction and intermediate powder respectively, place them in a stoppered conical flask, add 20 ml of water to dissolve them, shake and extract with n-butanol twice, 20 ml each time, combine the n-butanol solutions, evaporate to dryness, add 2 ml of 50% methanol to dissolve the residue, and use it as the test solution.

[0095] (2) Accurately pipette 1 μl of the reference substance solution, the reference medicinal material solution and the test solution prepared in step (1), inject them into an ultra-high performance liquid chromatograph, perform liquid chromatography analysis, and obtain a corresponding ultra-high performance liquid chromatogram.

[0096] The chromatographic condition parameters are as follows: GOLD aQ (Thermo, 2.1×150 mm, 1.9 μm) as the chromatographic column; methanol as the mobile phase A, 0.1% formic acid solution as the mobile phase B, and gradient elution according to the provisions in Table 1; the flow rate is 0.25 ml per minute; the column temperature is 25° C.; and the detection wavelength is 254 nm.

[0097] like Figure 4 As shown in the characteristic spectrum of the formula granules of Patrinia scabra, 14 characteristic peaks are presented in the chromatogram of the test solution, and the retention times of the 14 characteristic peaks in the chromatogram of the reference medicinal material reference material correspond to each other, among which peaks 1, 3, 7 and 10 should be consistent with the retention times of the protocatechuic acid reference material, danshen glycoside reference material, 3,4-O-dicaffeoylquinic acid reference material and 4,5-O-dicaffeoylquinic acid reference material respectively; the peak corresponding to the danshen glycoside reference material is the S peak, and the relative retention times of the remaining peaks and the S peak are calculated. The relative retention times should be within ±10% of the specified values, and the specified values ​​are: 0.55 (peak 2), 1.05 (peak 4), 1.07 (peak 5), 1.17 (peak 6), 1.82 (peak 8), 1.94 (peak 9), 2.34 (peak 11), 2.51 (peak 12), 2.54 (peak 13) and 2.61 (peak 14).

[0098] The characteristic spectrum of the Herba Patriniae Formula Granules was used to generate a reference characteristic spectrum ( Figure 5 ).

[0099] According to the test method of Herba Patriniae Formula Granules, Herba Patriniae Intermediates, Herba Patriniae Formula Granules, Herba Patriniae Standard Decoction, Herba Patriniae Decoction Pieces, and Herba Patriniae Materials were tested respectively. The test results of the test solution are shown in Tables 4 to 7 and Figures 6 to 9 .

[0100] Table 4 Determination results of samples of Patrinia scabra (Patrimonium wilfordii) formula granules (relative retention time)

[0101]

[0102]

[0103] Table 5 Determination results of standard decoction samples of Patrinia scabra (Patrimonium wilfordii) (relative retention time)

[0104]

[0105]

[0106]

[0107] Table 6 Determination results of samples of Patrinia scabra (Patrimonia chrysantha) (relative retention time)

[0108]

[0109]

[0110] Table 7 Determination results of Patrinia scabra (Patrimonium wilfordii) medicinal material samples (relative retention time)

[0111]

[0112]

[0113]

[0114] From the above test results, it can be seen that from a qualitative analysis point of view, the characteristic spectra of 3 batches of Herba Patriniae formula granules and 17 batches of Herba Patriniae standard decoctions, herbal slices and medicinal materials meet the requirements of the characteristic spectra in step (2).

[0115] Example 3 Characteristic Spectrum Methodology Study

[0116] 1. Exclusive inspection

[0117] The excipients added to the Herba Patriniae formula granules are maltodextrin, silicon dioxide and magnesium stearate. This experiment investigates the effect of negative samples lacking Herba Patriniae on Herba Patriniae formula granules. Take the negative sample lacking Herba Patriniae and prepare the negative sample solution according to the test sample preparation method. Inject 1 μl of the sample according to the above chromatographic conditions. The results are shown in Fig.10 .

[0118] The results showed that the solvents and excipients had no interference in the determination of the characteristic spectrum of the Patrinia scabra granules, and this method was specific for the determination of the Patrinia scabra granules.

[0119] 2. Overall inspection

[0120] Take an appropriate amount of Patrinia scabra (Painted with yellow flower) formula granules (batch number: KL1129), grind them into powder, take 0.3g, put them in a stoppered conical flask, add 20ml of water to dissolve, shake and extract with n-butanol twice, 20ml each time, combine the n-butanol solution, evaporate to dryness, add 2ml of 50% methanol to dissolve the residue, and use it as the test solution. Refer to the chromatographic conditions of Example 2, only change the elution gradient. Under the same chromatographic conditions, keep the elution gradient when the methanol ratio is the highest, and double the elution time. The results are shown in Fig.11 .

[0121] The results showed that no obvious chromatographic peaks were eluted after the original gradient elution, indicating that the chromatographic conditions basically met the principle of maximum information content.

[0122] 3. Precision inspection

[0123] Take Patrinia scabra (Painted Saposhnikovia divaricata) formula granules (batch number: KL1129) and prepare a test solution according to the test solution preparation method in Example 2. Inject 6 times continuously, 1 μl each time, record the retention time of its characteristic peak, and calculate the relative retention time according to the method of Example 2. The results are shown in Table 8.

[0124] Table 8 Precision test results (relative retention time)

[0125]

[0126]

[0127] The results showed that the RSD of the relative retention time of each characteristic peak was less than 1%, and the precision was good.

[0128] 4. Intermediate precision inspection

[0129] A sample of Patrinia scabra (Patrimonium wilfordii) formula granules (batch number: KL1129) was taken and prepared by two experimenters A and B according to the test solution preparation method determined in Example 2. 1 μl was injected at different times and on different instruments according to the chromatographic conditions of Example 2, and the retention time of the characteristic peak was recorded. The relative retention time was calculated. The results are shown in Table 9.

[0130] Table 9 Intermediate precision study (relative retention time)

[0131]

[0132]

[0133] The results showed that the RSD of the relative retention time of each characteristic peak was less than 3%, and the intermediate precision was good.

[0134] 5. Stability test

[0135] Take Patrinia scabra (Painted Saposhnikovia divaricata) formula granules (batch number: KL1129) and prepare a test solution according to the test solution preparation method determined in Example 2. Inject 1 μl at 0, 2, 4, 8, 12, 18, and 24 h, respectively, and detect according to the chromatographic conditions of Example 2. Record the retention time of its characteristic peak and calculate the relative retention time. The results are shown in Table 10.

[0136] Table 10 Stability study (relative retention time)

[0137]

[0138]

[0139] The results showed that the RSD of the relative retention time of each characteristic peak was less than 1%, and the test solution had good stability within 24 hours.

[0140] 6. Repeatability test

[0141] Take Patrinia scabra (Painted Saposhnikovia divaricata) formula granules (batch number: KL1129) in 6 parallel groups, prepare test solutions according to the test solution preparation method of Example 2, inject 1 μl of each solution, and detect according to the chromatographic conditions of Example 2, record the retention time of the characteristic peak, and calculate the relative retention time. The results are shown in Table 11.

[0142] Table 11 Repeatability test (relative retention time)

[0143]

[0144]

[0145] The results showed that the RSD of the relative retention time of each characteristic peak was less than 1%, and the repeatability of the method was good.

[0146] 7. Durability inspection

[0147] (1) Chromatographic column inspection

[0148] Take Patrinia chinensis (Patrimonium wilfordii) formula granules (batch number: KL1129), prepare the test solution according to the preparation method of the test solution in Example 2, and use GOLD aQ (Thermo, 2.1×150mm, 1.9μm); SB-C18 (Agilent, 2.1×150mm, 1.8μm); Syncronis C18 (Thermo, 2.1mm×100mm, 1.7μm) three different chromatographic columns, and other chromatographic conditions are the same as in Example 2, and the separation effect is investigated. The results are shown in Fig.12 , Table 12.

[0149] Table 12 Chromatographic column investigation (relative retention time)

[0150]

[0151]

[0152] The results showed that the Herba Patriniae samples could be well separated in the GOLD aQ (Thermo, 2.1×150mm, 1.9μm) column, and some chromatographic peaks were overlapped in the SB-C18 (Agilent, 2.1×150mm, 1.8μm) and Syncronis C18 (Thermo, 2.1mm×100mm, 1.7μm) columns. The subsequent studies in this experiment used the GOLD aQ (Thermo, 2.1×150mm, 1.9μm) column.

[0153] (2) Column temperature investigation

[0154] Take Patrinia chinensis (Patrimonium wilfordii) formula granules (batch number: KL1129), prepare the test solution according to the preparation method of the test solution in Example 2, use GOLD aQ (Thermo, 2.1×150mm, 1.9μm) chromatographic column, and examine the sample separation effect when the column temperature is 20℃, 25℃, and 30℃ respectively. Other chromatographic conditions are the same as in Example 2. The results are shown in Fig.13 , Table 13.

[0155] Table 13 Column temperature investigation (relative retention time)

[0156]

[0157]

[0158] The results show that when the column temperature is 20℃, peaks 4 and 5 form a peak envelope, and when the temperature is in the range of 25℃ to 30℃, the relative retention time of each characteristic peak is within the specified range, and a small column temperature change can meet the system durability requirements. The column temperature of 25℃ was selected for subsequent studies.

[0159] (3) Flow rate investigation

[0160] Take Patrinia scabra (Painted with yellow flower) formula granules (batch number: KL1129), prepare the test solution according to the preparation method of the test solution in Example 2, use GOLD aQ (Thermo, 2.1×150mm, 1.9μm) chromatographic column, and examine the sample separation effect at flow rates of 0.23ml / min, 0.25ml / min, and 0.27ml / min, respectively. Other chromatographic conditions are the same as in Example 2. The results are shown in Fig.14 , Table 14.

[0161] Table 14 Flow rate investigation (relative retention time)

[0162]

[0163] The results show that when the flow rate is in the range of 0.23-0.27 ml / min, the relative retention time of each characteristic peak is within the specified range, and a small flow rate change can meet the system durability requirements. The flow rate of 0.25 ml / min was selected for subsequent research.

[0164] Example 4 Identification study of different origins of Patrinia scabra

[0165] 1. Preparation of test solution

[0166] Take 0.3 g of the powder of the standard decoction of Patrinia lutea and the standard decoction of Patrinia dasyphylla, place it in a stoppered conical flask, add 20 ml of water to dissolve it, shake and extract it with n-butanol twice, 20 ml each time, combine the n-butanol liquids, evaporate to dryness, add 2 ml of 50% methanol solution to the residue to dissolve it, and use it as the test solution.

[0167] 2. Sample measurement and data analysis

[0168] 17 batches of standard decoction of Patrinia huanghua and 3 batches of standard decoction of Patrinia baihua were taken respectively, and sample test solutions were made according to the above-mentioned test solution preparation method. 1 μl was accurately pipetted and injected into an ultra high performance liquid chromatograph. According to the chromatographic conditions of Example 2, the characteristic spectra of the standard decoctions of Patrinia huanghua and Patrinia baihua were respectively determined, and the “Similarity Evaluation System of Chromatographic Fingerprints of Traditional Chinese Medicine (2012 Edition)” recommended by the State Pharmacopoeia Committee was used to generate a control characteristic spectrum, and then the control characteristic spectra of the two were imported into the “Similarity Evaluation System of Chromatographic Fingerprints of Traditional Chinese Medicine (2012 Edition)” for comparative analysis.

[0169] The results are as follows Fig.15 As shown, some characteristic peaks in the standard decoction of Huanghua Patriniae were not detected in Baihua Patriniae, and only 7 common characteristic peaks in Huanghua Patriniae were presented, among which peaks 5, 8 and 12 only existed in some batches of Baihua Patriniae; while peaks 9, 11, 13 and 14 were not detected in the standard decoction of Baihua Patriniae.

[0170] The differences between the quasi-decoctions of Radix Patriniae Sinensis and Radix Patriniae Albizia were further analyzed by relative peak area. Peak 3 was used as the reference peak to compare the relative peak area differences of the characteristic peaks in the standard decoctions of the two. The results are shown in Tables 15 and 16.

[0171] Table 15 Relative peak area range of standard decoction samples of Patrinia scabra and Patrinia dasyphylla

[0172]

[0173]

[0174] Table 16 Relative peak areas of standard decoction samples of Patrinia scabra and Patrinia dasyphylla

[0175]

[0176]

[0177] The results showed that peak 11 was not detected in the standard decoction of Phellodendron chinense. According to the relative peak area range, the minimum value was taken as 40% lower than the relative peak area lower limit, and the relative peak area of ​​peak 11 was not less than 0.24. Peak 8 was relatively small in the standard decoction of Phellodendron chinense. According to the relative peak area range, the minimum value was taken as 40% lower than the relative peak area lower limit, and the relative peak area of ​​peak 8 was not less than 0.30. In this way, the Phellodendron chinense samples with different origins of Patrinia chinensis can be effectively distinguished.

[0178] Example 5 Study on identification of authentic Patrinia scabra

[0179] 1. Preparation of test solution

[0180] Take 0.3 g of the powder of this product, place it in a stoppered conical flask, add 20 ml of water to dissolve it, shake and extract it twice with n-butanol, 20 ml each time, combine the n-butanol liquids, evaporate to dryness, add 2 ml of 50% methanol to dissolve the residue, and use it as the test solution.

[0181] 2. Sample measurement and data analysis

[0182] 17 batches of standard decoction of Patrinia scabra, 3 batches of standard decoction of Mutouhui (counterfeit Patrinia scabra), and 3 batches of standard decoction of Cyperus rotundus (counterfeit Patrinia scabra) were taken respectively, and sample test solutions were made according to the above-mentioned test solution preparation method. 1 μl was accurately pipetted and injected into an ultra high performance liquid chromatograph. According to the chromatographic conditions of Example 2, the characteristic spectra of the standard decoction of Patrinia scabra and its counterfeit were respectively determined, and the “Similarity Evaluation System of Chromatographic Fingerprint of Traditional Chinese Medicine (2012 Edition)” recommended by the State Pharmacopoeia Committee was used to generate a control characteristic spectrum, and then the control characteristic spectrum of Patrinia scabra and its counterfeit was imported into the “Similarity Evaluation System of Chromatographic Fingerprint of Traditional Chinese Medicine (2012 Edition)” for comparative analysis.

[0183] (1) Patrinia scabra and Herba Lycopodii

[0184] The results are as follows Fig.15 As shown in the figure, the number of characteristic peaks of the standard decoction of Mutouhui and the standard decoction of Patrinia scabra is different. The standard decoction of Patrinia scabra has 14 common characteristic peaks, while the standard decoction of Mutouhui only presents 8 common characteristic peaks in the standard decoction of Patrinia scabra, of which peak 12 only exists in some batches of Mutouhui; in addition, peak A is a new characteristic peak of Mutouhui, which has poor separation in Patrinia scabra and low response in most batches, and is not determined as a characteristic peak of Patrinia scabra; therefore, the established characteristic spectrum can effectively distinguish Patrinia scabra and Mutouhui standard decoction.

[0185] (2) Patrinia scabra and Cyperus rotundus

[0186] The results are as follows Fig.16 As shown in the figure, there is also a difference in the number of characteristic peaks between the standard decoction of Herba Patriniae and the standard decoction of Herba Patriniae. The standard decoction of Herba Patriniae has 14 common characteristic peaks, while the standard decoction of Herba Patriniae only presents 7 common characteristic peaks in the standard decoction of Herba Patriniae; in addition, peaks B to E are newly added common characteristic peaks of Herba Patriniae, which do not exist in Herba Patriniae; therefore, the standard decoctions of Herba Patriniae and Herba Patriniae can be effectively distinguished by the presence or absence of peaks B to E.

[0187] The differences between the standard decoction of Patrinia salsa and Mutouhui were further analyzed by relative peak area. Peak 3 was used as the reference peak to compare the relative peak area differences of the characteristic peaks in the standard decoction of Patrinia salsa and Mutouhui. The results are shown in Tables 17 and 18.

[0188] Table 17 Relative peak area range of Patrinia scabra and standard decoction samples of Mutouhui

[0189]

[0190]

[0191] Table 18 Relative peak areas of Patrinia scabra and standard decoction samples of Mutouhui

[0192]

[0193]

[0194] The results showed that peak 11 in the standard decoction of Patrinia salsa was not detected in the standard decoctions of Mutouhui and Pinellia ternata. According to the relative peak area range, the minimum value was taken as 40% lower than the relative peak area lower limit, and the relative peak area of ​​peak 11 was not less than 0.24. Peak 8 was smaller in the standard decoction of Patrinia dasyphylla. According to the relative peak area range, the minimum value was taken as 40% lower than the relative peak area lower limit, and the relative peak area of ​​peak 8 was not less than 0.30. At the same time, neither peak 11 nor peak 8 was detected as Mutouhui, and Pinellia ternata was not detected in peak 3. In this way, the fake Patrinia salsa products Mutouhui and Pinellia ternata can be effectively distinguished.

Claims

1. A method for constructing a characteristic map of Patrinia scabra, characterized in that: The following steps are involved: (1) Take the powder of Patrinia lappa herb, intermediate, decoction piece, standard decoction or its formula granules respectively, add water to dissolve, extract with n-butanol, evaporate the extract to dryness, and add methanol aqueous solution to dissolve the residue to prepare the test solution; (2) respectively taking protocatechuic acid, dianthus glycoside, 3,4-O-dicaffeoylquinic acid, and 4,5-O-dicaffeoylquinic acid, and adding a solvent to prepare a reference substance solution; (3) Inject the test solution and the reference solution into an ultra-high performance liquid chromatograph to obtain a characteristic spectrum; The characteristic spectrum has 14 peaks in total. Protocatechuic acid is taken as a reference substance, and peak 1 is the chromatographic peak of protocatechuic acid; dapoxetine is taken as a reference substance, and peak 3 is the chromatographic peak of dapoxetine; 3,4-O-dicaffeoylquinic acid is taken as a reference substance, and peak 7 is the chromatographic peak of 3,4-O-dicaffeoylquinic acid; 4,5-O-dicaffeoylquinic acid is taken as a reference substance, and peak 10 is the chromatographic peak of 4,5-O-dicaffeoylquinic acid; the peak corresponding to the dapoxetine reference substance is the S peak, and the relative retention time of the remaining peaks and the S peak is calculated; the relative retention time should be within ±10% of the specified value, and the specified values ​​of peak 2, peaks 4 to 6, peak 8, peak 9, and peaks 11 to 14 are: 0.55, 1.05, 1.07, 1.17, 1.82, 1.94, 2.34, 2.51, 2.54, and 2.61, respectively.

2. The construction method according to claim 1, characterized in that: The number of extractions in step (1) is 1 to 3 times, and the extraction method is shaking.

3. The construction method according to claim 1, characterized in that: In step (1), the concentration of the protocatechuic acid reference substance solution is 15 to 40 μg / ml; the concentration of the glycoside reference substance solution is 100 to 200 μg / ml; the concentration of the 3,4-O-dicaffeoylquinic acid reference substance solution is 15 to 40 μg / ml; and the concentration of the 4,5-O-dicaffeoylquinic acid reference substance solution is 15 to 40 μg / ml.

4. The construction method according to claim 1, characterized in that: The chromatographic conditions of the ultra-high performance liquid chromatography described in step (3) are as follows: GOLD aQ as the chromatographic column; methanol as the mobile phase A and 0.1% formic acid solution as the mobile phase B; a flow rate of 0.23-0.27 ml / min; a column temperature of 25-30° C.; a detection wavelength of 254 nm; a gradient elution of 0-3 min, a mobile phase A volume fraction of 10%, and a mobile phase B volume fraction of 90%; 3-22 min, the volume fraction of the mobile phase A changes from 10 to 26%, and the volume fraction of the mobile phase B changes from 90 to 74%; 22-34 min, the volume fraction of the mobile phase A changes from 26 to 36%, and the volume fraction of the mobile phase B changes from 74 to 64%; 34-55 min, the volume fraction of the mobile phase A changes from 36 to 56%, and the volume fraction of the mobile phase B changes from 64 to 44; 55-58 min, the volume fraction of the mobile phase A is 56%, and the volume fraction of the mobile phase B is 44%.

5. A method for constructing a reference map of Patrinia scabra, characterized in that: The following steps are involved: (1) preparing multiple batches of Patrinia chinensis herb test solution, Patrinia chinensis intermediate test solution, Patrinia chinensis decoction piece test solution, Patrinia chinensis standard decoction test solution or Patrinia chinensis formula granule test solution; (2) The method for constructing a characteristic spectrum of Patrinia chinensis according to claim 1 is used to detect the test solution respectively to obtain a characteristic spectrum of Patrinia chinensis medicinal materials, a characteristic spectrum of intermediates, a characteristic spectrum of decoction pieces, a characteristic spectrum of standard decoctions, and a characteristic spectrum of formula granules; (3) The obtained characteristic spectrum is introduced into the Chinese medicine chromatographic fingerprint spectrum similarity evaluation system to establish a reference spectrum of Patrinia scabra medicinal materials, intermediates, decoction pieces, standard decoctions or their formula granules.

6. A method for distinguishing Patrinia lutea from Patrinia odorata, characterized in that: The following steps are involved: (1) Take the powder of the medicinal material, intermediate, decoction piece, standard decoction or its formula granules of Patrinia chrysantha or Patrinia dasyphylla respectively, add water to dissolve it, extract it with n-butanol, evaporate the extract to dryness, and add methanol-water solution to dissolve the residue to prepare the test solution; (2) respectively taking protocatechuic acid, dianthus glycoside, 3,4-O-dicaffeoylquinic acid, and 4,5-O-dicaffeoylquinic acid, and adding a solvent to prepare a reference substance solution; (3) The test sample solution and the reference sample solution were injected into an ultra-high performance liquid chromatography to obtain characteristic spectra; by analyzing the relative peak areas of peaks 8 and 11 relative to peak 3 in Patrinia chinensis, Patrinia chrysantha and Patrinia leucoderma can be distinguished; when the relative peak area of ​​peak 11 is greater than 0.24 and the relative peak area of ​​peak 8 is greater than 0.30, the test sample is Patrinia chrysantha; when peak 11 is less than 0.24 and peak 8 is less than 0.30, the test sample is Patrinia leucoderma.

7. The method according to claim 6, characterized in that: The chromatographic conditions of the ultra-high performance liquid chromatography are as follows: GOLDaQ is used as a chromatographic column; methanol is used as a mobile phase A, and a 0.1% formic acid solution is used as a mobile phase B; the flow rate is 0.23-0.27 ml / min; the column temperature is 25-30° C.; the detection wavelength is 254 nm; the gradient elution is 0-3 min, the volume fraction of the mobile phase A is 10%, and the volume fraction of the mobile phase B is 90%; 3-22 min, the volume fraction of the mobile phase A changes from 10 to 26%, and the volume fraction of the mobile phase B changes from 90 to 74%; 22-34 min, the volume fraction of the mobile phase A changes from 26 to 36%, and the volume fraction of the mobile phase B changes from 74 to 64%; 34-55 min, the volume fraction of the mobile phase A changes from 36 to 56%, and the volume fraction of the mobile phase B changes from 64 to 44; 55-58 min, the volume fraction of the mobile phase A is 56%, and the volume fraction of the mobile phase B is 44%.

8. A method for distinguishing Patrinia chrysantha from counterfeit products, characterized in that: The following steps are involved: (1) Take the powder of Patrinia chrysanthemi or counterfeit medicinal materials, intermediates, decoction pieces, standard decoction or its formula granules respectively, add water to dissolve, extract with n-butanol, evaporate the extract to dryness, and add methanol-water solution to dissolve the residue as the test solution; (2) respectively taking protocatechuic acid, dianthus glycoside, 3,4-O-dicaffeoylquinic acid, and 4,5-O-dicaffeoylquinic acid, and adding a solvent to prepare a reference substance solution; (3) The test sample solution and the reference sample solution were injected into an ultra-high performance liquid chromatography to obtain characteristic spectra; by analyzing the relative peak areas of peaks 8 and 11 in Patrinia scabra relative to peak 3, Patrinia scabra and its counterfeits, Herba Epimedii and Herba Cynoglossi, could be distinguished; when the relative peak area of ​​peak 11 was greater than 0.24 and the relative peak area of ​​peak 8 was greater than 0.30, the test sample was Patrinia scabra; when peaks 11 and 8 were not detected, the test sample was Herba Cynoglossi; when peak 3 was not detected, the test sample was Herba Cynoglossi.

9. The method according to claim 8, characterized in that: The chromatographic conditions of the ultra-high performance liquid chromatography are as follows: GOLDaQ is used as a chromatographic column; methanol is used as a mobile phase A, and a 0.1% formic acid solution is used as a mobile phase B; the flow rate is 0.23-0.27 ml / min; the column temperature is 25-30° C.; the detection wavelength is 254 nm; the gradient elution is 0-3 min, the volume fraction of the mobile phase A is 10%, and the volume fraction of the mobile phase B is 90%; 3-22 min, the volume fraction of the mobile phase A changes from 10 to 26%, and the volume fraction of the mobile phase B changes from 90 to 74%; 22-34 min, the volume fraction of the mobile phase A changes from 26 to 36%, and the volume fraction of the mobile phase B changes from 74 to 64%; 34-55 min, the volume fraction of the mobile phase A changes from 36 to 56%, and the volume fraction of the mobile phase B changes from 64 to 44; 55-58 min, the volume fraction of the mobile phase A is 56%, and the volume fraction of the mobile phase B is 44%.

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